In Vitro and In Silico Potential Inhibitory Effects of New Biflavonoids from Ochna rhizomatosa on HIV-1 Integrase and Plasmodium falciparum.

The aim of this study was to identify bioactive secondary metabolites from Ochna rhizomatosa with potential inhibitory effects against HIV and Plasmodium falciparum. A phytochemical study of O. rhizomatosa root barks resulted in the identification of three new biflavonoids (1-3), along with four known ones (4-7). Compound 7 (Gerontoisoflavone A) was a single flavonoid present in the rootbark of the plant and was used as a reference. Compound 1 (IC50 = 0.047 µM) was the only one with a noteworthy inhibitory effect against HIV-1 integrase in vitro. Chicoric acid (IC50 = 0.006 µM), a pure competitive inhibitor of HIV-1 integrase, was used as control. Compound 2 exhibited the highest antiplasmodial activity (IC50 = 4.60 µM) against the chloroquine-sensitive strain of Plasmodium falciparum NF54. Computational molecular docking revealed that compounds 1 and 2 had the highest binding score (-121.8 and -131.88 Kcal/mol, respectively) in comparison to chicoric acid and Dolutegravir (-116 and -100 Kcal/mol, respectively), towards integrase receptor (PDB:3LPT). As far as Plasmodium-6 cysteine s48/45 domain inhibition is concerned, compounds 1 and 2 showed the highest binding scores in comparison to chloroquine, urging the analysis of these compounds in vivo for disease treatment. These results confirm the potential inhibitory effect of compounds 1 and 2 for HIV and malaria treatment. Therefore, our future investigation to find inhibitors of these receptors in vivo could be an effective strategy for developing new drugs.

Khaya grandifoliola active fraction as a source of therapeutic compounds for Alzheimer's disease treatment: In silico validation of identified compounds.

Overproduction of Nitric oxide (NO) and many other pro-inflammatory mediators are responsible for many pathological disorders in humans, including Alzheimer's disease (AD). In this study, active fractions isolated from Khaya grandifoliola (Kg) were screened for their inhibitory activities against NO production in lipopolysaccharide (LPS)-activated microglia. Among the 5 fractions tested, Kg25 was the most active and showed potent inhibitory activity towards NO production. The fraction further showed inhibitory effect on iNOS's mRNA expression and other major pro-inflammatory cytokines including TNFα and IL1-ß. Study of the effect of Kg25 on p38MAPKinase and JNK3 showed that the fraction inhibits these signaling pathways known to be involved in cell inflammatory pathways. These observations were confirmed at the protein level with Kg25 inhibiting iNOS and p38MAPK protein expressions in N9 cells. Analysis of Kg25 composition by HPLC identified 3 main compounds, namely: 6 phenyl, 4-(1`oxyehylphenyl) hexane, Carbamic acid, (4-methly-1-phenyl)-1, phenyl, and Benzene, 1 1`-(oxydiethylidene) bis. The above mentionned compounds were further analyzed for their bioactivity against the p38MAPKinase and iNOS receptors using molecular docking. MolDock results showed that 1-phenylethyl N-(4-methylphenyl)carbamate (compound 2) possesses the highest binding affinity (for iNOS); and 1-(1-phenylethoxy)ethylbenzene (compound 3) (for pMAPK) respectively and both compounds interact well with the active site residues. Hence, these compounds could be considered as scaffolds for further development of lead- drugs targeting neuroinflammation in AD.

Inhibition of CYP2E1 and activation of Nrf2 signaling pathways by a fraction from Entada africana alleviate carbon tetrachloride-induced hepatotoxicity.

Entada africana is used in non-conventional medicine for the management of liver ailments. A fraction, designated EaF10 (methylene chloride/methanol 90:10, v/v) with promising hepatoprotective activity has been isolated. Since the mechanisms underlying EaF10 hepatoprotective action remain unknown, this study was undertaken to investigate the anti-hepatotoxic mechanism of the fraction against carbon tetrachloride (CCl4)-induced hepatotoxicity and its antioxidant properties. Antioxidant activities of EaF10 were assessed through four chemical antioxidant assays and its anti-hepatotoxic effect evaluated in vivo and in vitro by post-treatment (25 or 100 mg/Kg) or co-treatment (6.25-100 µg/mL) in CCl4-intoxicated mice and normal human liver cells line L-02 hepatocytes respectively; and biochemical and molecular parameters assessed respectively by spectrophotometry, and by quantitative real-time polymerase chain reaction and western blot analysis. EaF10 exhibited strong antioxidant activities correlated with its polyphenol content. Serum levels of alanine/aspartate aminotransferase (AST/ALT) and nitrite oxide, liver contents of glutathione (GSH) protein carbonylation and malondialdehyde (MDA), liver activities of catalase (CAT), glutathione-S-transferase (GST) and superoxide dismutase (SOD) and cell viability showed the anti-hepatotoxic effect of EaF10, supported by histopathological observations. The fraction decreased the protein level of Cytochrome P450 2E1 (CYP2E1) and Kelch-like ECH-associated protein-1 (Keap-1), induced nuclear translocation of Nuclear factor-erythroid 2-related factor-2 (Nrf2) coupled to an increase of the mRNA levels of CAT, SOD1 and GST in CCl4-intoxicated L-02 hepatocytes. These findings evidenced that the studied plant fraction possesses a strong antioxidant capacity and prevents CCl4-induced hepatotoxicity, likely through inhibition of CYP2E1 and activation of the Nrf2 signaling pathway.

Natural compounds flavonoids as modulators of inflammasomes in chronic diseases.

The use of dietary or medicinal plant based natural compounds to disease treatment has become a unique trend in clinical research. Flavonoids, a group of polyphenolic compounds have drawn significant attention due to their modulatory effects on inflammasomes associated with the initiation and progression of chronic disorders including metabolic, neurodegenerative diseases and cancer. In this article, the role of most commonly studied natural flavonoids with their disease-specific impact via inflammasomes as a potential molecular target has been described. Since the role of inflammation is evident in multiple diseases, flavonoids may serve as a promising tool in drug discovery for the intervention of chronic diseases by manipulating the status of inflammation via inflammasome targeting.

Withaferin A modulates AIM2 inflammasome and caspase-1 expression in THP-1 polarized macrophages.

Inflammasomes are cytoplasmic protein complexes that regulate the secretion of pro-inflammatory cytokines including IL-1ß and IL18, thereby playing a crucial role in inflammatory and chronic diseases. Plant compound Withaferin A (WFA) has been demonstrated to possess numerous biological activities including anti-inflammatory and anti-cancer effects. However, the effect of WFA on macrophage polarization and inflammasome expression in polarized macrophages has not been documented. In this study, cultured THP-1 macrophages were polarized into M1/M2 phenotypes. Subsequently, macrophage characterization was tested for M1 markers (CXCL10 and CXCL9) and M2 markers (CCL20 and CCL13). NOD-like receptor protein 3 (NLRP3) and Absent in melanoma (AIM2) inflammasome gene and protein expressions were measured by RTqPCR and Western blot respectively. Colocalization of both proteins in polarized macrophages was analyzed by immunofluorescence. Our results show that M1 polarized macrophages express elevated NLRP3 and AIM2 gene expressions. Furthermore, WFA treatment stimulated AIM2 and caspase-1 protein expression in M2W macrophages in comparison to M2 cells. ELISA analysis of the cell culture supernatant showed that WFA treatment of M2 macrophages inhibited the secretion of TGF-ß in comparison to M1. Immunofluorescence studies showed NLRP3/ASC colocalized in the cytoplasm in M1 macrophages, which was not the case in M2 and M2W cells. AIM2/ASC were found colocalized in M1 and M2W cells, indicating an activation of inflammasome. These results provide basis for better understanding the effect of WFA in inflammatory diseases and some cancers by modulating macrophage polarization and inflammasome activation.

Amelioration of Behavioral Impairments and Neuropathology by Antiepileptic Drug Topiramate in a Transgenic Alzheimer's Disease Model Mice, APP/PS1.

Alzheimer's disease (AD) is a neurodegenerative disease that is the main cause of dementia in the elderly. The aggregation of ß-amyloid peptides is one of the characterizing pathological changes of AD. Topiramate is an antiepileptic drug, which in addition, is used in the treatment of many neuropsychiatric disorders. In this study, the therapeutic effects of topiramate were investigated in a transgenic mouse model of cerebral amyloidosis (APP/PS1 mice). Before, during, and after topiramate treatment, behavioral tests were performed. Following a treatment period of 21 days, topiramate significantly ameliorated deficits in nest-constructing capability as well as in social interaction. Thereafter, brain sections of mice were analyzed, and a significant attenuation of microglial activation as well as ß-amyloid deposition was observed in sections from topiramate-treated APP/PS1 mice. Therefore, topiramate could be considered as a promising drug in the treatment of human AD.

Protective Effects of Forskolin on Behavioral Deficits and Neuropathological Changes in a Mouse Model of Cerebral Amyloidosis.

The production of amyloid-ß peptides in the brains of patients with Alzheimer disease (AD) may contribute to memory loss and impairments in social behavior. Here, an efficient adenylate cyclase activator, forskolin, was orally administered by gavage (100 mg/kg body weight) to 5-month-old transgenic APP/PS1 mice, which serve as an animal model of cerebral amyloidosis. Analyses of nest construction, sociability, and immunohistochemical features were used to determine the effects of forskolin treatment. After a relatively short term of treatment (10 days), forskolin-treated transgenic mice showed restored nest construction ability (p < 0.05) and their sociability (p < 0.01). There was a reduction of Aß plaque deposition in the cortex and in the hippocampus. Furthermore, expression of transforming growth factor ß, glial fibrillary acidic protein, and Iba-1 in the cortex was reduced in the forskolin-treated group, suggesting regulation of the inflammatory response mediated by activated microglia and astrocytes in the brains of the APP/PS1 mice (p < 0.01). Taken together, these findings suggest that forskolin shows neuroprotective effects in APP/PS1 Tg mice and may be a promising drug in the treatment of patients with AD.

Molecular Insight in the Multifunctional Effects of Oridonin.

Oridonin has attracted considerable attention in the last decade because of its anti-cancer pharmacological properties. This ent-kaurane diterpenoid, isolated from the Chinese herb Rabdosia rubescens and some related species, has demonstrated great potential in the treatment profile of many diseases by exerting anti-tumor, anti-inflammatory, pro-apoptotic, and neurological effects. Unfortunately, the mechanisms via which oridonin exerts these effects remain poorly understood. This review provides an overview of the multifunctional effects of oridonin as well as the reasons for its potential for investigations in the treatment of many diseases other than cancer.

Entada africana fraction CH2Cl2/MEOH 5% inhibits inducible nitric oxide synthase and pro-inflammatory cytokines gene expression induced by lipopolysaccharide in microglia.

BACKGROUND: Inflammatory response in the CNS mediated by microglia cells play an important role in host defense and is implicated in the pathology of neurodegenerative diseases. We investigated the capacity of Entada africana to protect microglia from inflammatory insults by exploring the effect of the CH2Cl2/MEOH 5% fraction (Ea5) on pro-inflammatory cytokines mRNA expression. Finally, we studied the effect of Ea5 on the inhibition of p38 MAPK Kinase. The results were compared to those obtained with Baicalin, a well reported anti-inflammatory flavonoid. METHODS: Barks from E. africana were harvested in 2010, in the west region of Cameroon. A crude extract was prepared using CH2Cl2/MEOH 1:1 V/V. The crude extract obtained was further fractionated by flash chromatography. A mouse microglia cell line (N9) was stimulated by LPS with or without different concentrations of Baicalin and Ea5. The release of NO was evaluated using the Griess method. The expression of pro-inflammatory cytokines mRNA (TNFα, IL-1ß, IL-6) and iNOS/NO were measured by RT- PCR. The inhibition of p38 MAPK Kinase was assessed using ELISA. RESULTS: We found that Ea5, as well as Baicalin inhibited LPS-induced NO production in a dose dependent manner. Ea5 was most active in term of NO inhibition (87.07%), in comparison to Baicalin (70.85%). The expression of TNFα, IL-1ß, IL-6 and iNOS was strongly suppressed by Ea5 in microglia. Ea5 also inhibited the activity of p38MAPK Kinase, up to 30% for the concentrations tested, whereas a prominent inhibition was obtained with Baicalin. CONCLUSION: These results suggest that E. africana may contain promising compounds useful for the treatment of diseases cause by over-activation of microglia such as Alzheimer disease and other neurological diseases.